The invention relates to a luminescence-optical method for qualitative and quantitative determination of at least one analyte and/or component of a liquid measuring medium containing a chromophore (or a luminophore) which is directly or indirectly responsive to the component to be determined by changing its absorption spectrum, and a luminophore which is not responsive to the component to be determined, where there is at least partial overlap between the emission spectrum of the luminophore and the absorption spectrum of the chromophore, and where the nonradiative energy transfer between luminophore and chromophore produces a measurable change in at least one luminescence characteristic of the luminophore. That principle is known as the so-called FRET-principle.
The invention further relates to a reagent for quantitative determination of at least one analyte and/or component of a gaseous or liquid measuring medium containing a chromophore (or a luminophore) which is directly or indirectly responsive to the component to be determined by changing its absorption spectrum, and a luminophore which is not responsive to the component to be determined, where there is at least partial overlap between the emission spectrum of the luminophore and the absorption spectrum of the chromophore, and where the nonradiative energy transfer between luminophore and chromophore produces a measurable change in at least one luminescence characteristic of the luminophore.
In the following, luminophores are understood as dyes which emit phosphorescence or fluorescence radiation upon suitable excitation. The absorption spectrum of the chromophore is influenced either directly by the component to be measured or indirectly by a chemical reaction product of the component to be measured. The term “quantitative determinations of a chemical component” refers to the determination of concentration and activity as well as gas partial pressure, the values of at least one luminescence characteristic of the luminophore being used to infer the measured quantity.
A method and a sensor in which pH- and cation-sensitive chromophores (acceptor) are attached, preferably covalently, to a luminophore (donor) are known from U.S. Pat. No. 5,232,858. The pH-value and/or concentration of the cation to be determined in the measuring medium is derived from the luminescence decay time of the luminophore.
As far as the state of the art is concerned, U.S. Pat. No. 5,648,269 is also to be mentioned. This document suggests the application of the apparent luminescence decay time of the luminophore for determining the measured quantity. With luminophores with one decay time component, the apparent luminescence decay time is identical with the effective decay time. With luminophores with several decay time components, it is easier to evaluate the apparent decay time, however—in particular with systems that are not robust—there is the drawback of increasing errors.
Luminescence decay times may be obtained by means of phase-modulation or time-resolved luminescence measuring techniques, respectively.
A similar method is known from EP-A-0 214 768. Therein, the concentration of the parameter to be determined in the measuring medium is inferred from the luminescence intensity measured.
The rate of nonradiative energy transfer from donor to acceptor molecules depends on the spatial proximity of the molecules of the two substances. The transfer rate kT(r) is extremely responsive to the spatial distance r between donor and acceptor and decays with the sixth power of the distance
                    k        T            ⁡              (        r        )              =                  1                  τ          D                    ⁢                        (                                    R              O                        r                    )                6              ,whereby τD indicates the luminescence decay time of the donor in absence of the acceptor and RO indicates the characteristic Förster distance. The latter is that donor-acceptor distance in which a 50% efficiency of the energy transfer is provided. Depending on the respective donor-acceptor pair, RO is between 2 and 9 nm.
Due to the nonradiative energy transfer from donor to acceptor molecules, the macroscopically determinable values of the luminescence-optical parameters (luminescence quantum efficiency, luminescence intensity, luminescence decay time) of the luminophore will undergo a particularly efficient change if a substantial number of molecules of the two substances are brought into close spatial contact with each other.
To obtain close spatial contact, U.S. Pat. No. 5,232,858 proposes a covalent bonding of donor and acceptor molecules. In EP-A-0 214 768 individual donor and acceptor molecules are covalently attached to the surface of a joint substrate, such as glass.
The covalent bonding of donor and acceptor molecules as described in U.S. Pat. No. 5,232,858 has the advantage that the mean spatial distance of donor/acceptor may be kept as constant as possible. However, it is a disadvantage that particularly great synthesis efforts are required to produce covalent bonds between desirable luminophores and suitable pH- or ion-sensitive chromophores.
Considerable efforts are also needed to covalently attach donor and acceptor molecules to the surface of a joint substrate (EP-A-0 214 768), which, above all, brings about the drawback that boundary surface phenomena impair the quality of the measured results.
Thus, in U.S. Pat. No. 5,942,189 and U.S. Pat. No. 6,046,055 it is suggested that the luminophore and the chromphore are ionic substances of differing electrical charges, which are present as ionic pairs in a matrix material that is permeable to the chemical component to be determined.
The use of long-lived luminophores (having luminescence decay times >100 ns, preferably >1 μs), such as exhibited, for example, by metal-ligand complexes, certain porphyrins and lanthanides, is of utmost importance to a general commercial application. Long-lived luminescence provided, the opto-electronic arrangements and components for the determination of the luminescance decay time and/or values to be derived therefrom (for example, mean luminescance decay time, phase angle) may be determined in a particularly inexpensive manner by means of phase or time-resolved methods.
However, the inventor of the present application has recognized that the above-mentioned, previously known methods bring about the mutual disadvantage that in particular the luminescence of long-lived luminophores is influenced by a number of components of the measuring medium. A known characteristic of such luminophores is the particularly great dependency of the luminescence characteristic on the O2 content of the sample. Consequently, such luminophores are thus often used for determining the O2 content (EP-A-0 907 074). When using those luminophores as donor dyes with sensors based upon the FRET-principle, it is thus necessary to exactly know or determine the O2 content of the measuring medium and to carry out appropriate adjustments. Examples of further substances having an influence on the luminescence quantum efficiency are amines and water. In the course of continuous measurements (monitoring), luminescence dyes may be completely or partially destroyed by the emerging singlet-O2. Accordingly, additives for limiting that process were suggested. However, a general drawback of known, advantageous luminescence dyes is the luminescence characteristics' sensitivity to minor changes of the chemical-physical microenvironment, caused by any components of the sample, in particular water. In case of sample media of unknown and/or variable chemical or biochemical compositions, that leads to a significant limitation of the measuring accuracy. For example, in medical diagnostics, reproducibilities of +/−5 milli-pH are expected in the field of blood-pH determination.
It is the object of the invention to improve luminescence-optical determination methods based upon the FRET-principle, which are based upon a luminophore (donor) and a chromphore (acceptor, indicator) reversibly binding the substance (analyte) to be determined or its reaction products, with regard to the susceptance to failure caused by components of the sample to be measured. Furthermore, particularly great chemical synthesis steps needed to obtain the spatial proximity of a substantial number of acceptor molecules and shielded donor molecules are to be avoided.